Specific incorporation of selenium into 5-methylaminomethyl-2- selenouridine residues of lysine-, glutamate-, and glutamine-accepting TRNAS occurs in several bacterial species. In this process, the sulfur of the corresponding 2-thiouridine residue in these TRNAS is replaced with selenium. This replacement reaction is inactivated by mutation of the selD gene in Escherichia coli, referred to as selA1 in Salmonella typhimurium. We have reported earlier that the selD gene product, selenophosphate synthetase, catalyzes the formation of selenophosphate from selenide and ATP and that supplementation of the selA1 mutant cell preparation with the purified enzyme, ATP, and selenide restored synthesis of seleno-TRNAS. In our present work we found that activation of the selA1 mutant cell preparation can be achieved by addition of purified selenophosphate alone. The amount of 75Se incorporated into TRNAS from added [75Se]selenophosphate was similar to that incorporated when mutant enzyme preparations were supplemented with [75Se]selenide ATP and selenophosphate synthetase. Furthermore, addition of authentic non- radiolabeled selenophosphate to the reaction mixture decreased the amount of 75Se incorporated into TRNAS in a dose-dependent manner. In this analysis [75Se]seleno-phosphate was generated in situ by selenophosphate synthetase from [75Se]selenide and ATP. In a control experiment with [75Se]selenophosphate as substrate, non-labeled selenide did not affect the incorporation of 75Se into TRNAS indicating that seleno-phosphate is the actual Se donor for the selenation reaction. The replacement of sulfur in the TRNAS with selenium requires, in addition to selenophosphate, at least one enzyme in the crude mutant preparations. Added beta, gamma-methylene ATP did not inhibit and no ATP requirement was observed in this system suggesting a direct attack of selenophosphate on the 2-thio moiety of the uridine residue in the "wobble position" of TRNAS.